Screening active compound from Sanguisorba officinalis on induction of Leukocyte production via CD38 protein targeting in cancer therapy-induced Leukopenia

Abstract

Ovarian cancer is a malignant gynecological neoplasm, causing a high mortality rate and relapse rate for women. The focus of this study was on platinum-sensitive recurrent ovarian cancer (PSROC), one of the most prevalent types of recurrent ovarian cancer for which chemotherapy is still the primary choice of treatment. The guidelines have proposed some treatment strategies for PSROC, but the effectiveness of these might vary from person to person. Meanwhile, chemotherapy always causes serious leukopenia or neutropenia, severely hampering cancer treatment. Although G-CSF is widely accepted for leukopenia, it does not reduce the incidence of infection-related illnesses in leukopenia patients. Natural products and traditional herbal medicines can provide novel options for leukopenia treatment, one of these is Sanguisorba officinalis (S. officinalis) which is used in Chinese traditional clinics. However, the complex components and the uncertain mechanism hindered the further clinical use of S. officinalis. Evidence has shown that cluster of differentiation 38 (CD38) might possess advantages in improving leukopenia and the antibacterial ability of myeloid cells through the activation of ADP-ribosyl cyclase and the associated cascades. In this study, to seek the optimal therapeutic schedule for individualized PSROC, a Bayesian network meta-analysis was performed to compare the therapeutic effects and toxicity of different treatment options by indirect or direct evidence. To confirm whether CD38 represented a viable therapeutic target of leukopenia, a CD38 receptor-based pharmacophore virtual screening was first established to find novel CD38 agonists from S. officinalis and investigate bioactivities of the compounds on leukopenia. The results declared that the carboplatin-pegylated liposomal doxorubicin-bevacizumab combination provided the greatest progression-free survival (PFS) (hazard ratio [HR] 0.59, 95% credible interval [CI] is between 0.51and 0.68) in the initial stage of chemotherapy. The carboplatin- paclitaxel-bevacizumab combination had the best overall survival (OS) (HR 1.22, 95% CI 1.09 to 1.35), and objective response rate (ORR) (odds ratio 1.22, 95% CI 1.09 to 1.35). The maintenance stage analysis suggested that platinum-based-poly (ADP-ribose) polymerase inhibitors (PARPi) (HR 0.66, 95% CI 0.59 to 0.74) and platinum-based-bevacizumab (HR 0.64, 95% CI 0.55 to 0.75) had priority in patients with BRCA wide-type, but only platinum-based-PARPi was suitable for BRCA mutation patients. The safety evaluation showed that bevacizumab addition therapy and PARPi addition therapy resulted in a significant discontinuation rate and adverse events of grade three or higher, with thrombocytopenia and neutropenia grade ≥3 being more pronounced. Leukopenia potentially develops into a life-threatening disease in cancer patients, and effective drug treatments are still lacking in clinical practice. Thus, a pharmacophore generation algorithm based on the CD38 receptor was constructed for a novel drug discovery. Virtual screening and drug affinity responsive target stability (DARTS) test found three novel compounds (ziyuglycoside II, brevifolincarboxylic acid, and 3,4-dihydroxy-5-methoxybenzoic acid), which had a high binding affinity to the structure of CD38 protein. Likewise, enzymatic activity assay of ADP-ribosyl cyclase demonstrated the three selected monomers promoted cytoplasmic Ca2+ elevation and nicotinamide adenine dinucleotide (NAD+) degradation in NB4 cells (promyelocytic cell line), which indicated ziyuglycoside II, brevifolincarboxylic acid, and 3,4-dihydroxy-5-methoxybenzoic acid were the ADP-ribosyl cyclase agonists of CD38. The three ADP-ribosyl cyclase agonists in vitro confirmed that the differentiation of myeloid cells and the antibacterial function of NB4 cells were induced in an ADP-ribosyl cyclase-dependent manner. The ADP-ribosyl cyclase agonists were enriched in leukopoiesis, leukocyte immunity, and type II interferon or Interferon-gamma (IFN-γ) generation through network pharmacology prediction. The biological activity results of NB4 cells further demonstrated the induction of myeloid development and function by IFN-γ treatment. In addition, laser scanning confocal microscopy analysis, hematologic parameters measurement, and flow cytometry demonstrated that ADP-ribosyl cyclase agonists and IFN-γ successfully restored the number and bactericidal effect of myeloid cells in irradiation-induced leukopenia mice and a zebrafish model. Both in vitro and in vivo experiments revealed that ADP-ribosyl cyclase agonists could enhance the production and anti-infection effect of mature myeloid cells, which might be ascribed to ADP-ribosyl cyclase activity as well as IFN-γ synthesis. GO and KEGG tools revealed that the mechanism may be related to calcium signaling, positively regulated IFN-γ production, and myeloid formation. The incorporation of Ca2+ channel inhibitors caused the compounds to fail to induce IFN-γ expression and myeloid differentiation, which indicated that the activation of Ca2+ signaling mediates leukopoiesis in the ADP-ribosyl cyclase agonists treatment. Subsequently, Western blotting and immunofluorescence showed that the screen-out compounds could activate the calcineurin enzyme of NB4 cells and drive the nuclear factor of activated T cells 2 (NFAT2) expression and nuclear translocation. Likewise, the Ca2+ channel inhibitors, the calcineurin activity inhibitor suppressed the synthesis of IFN-γ and the myeloid differentiation in the NB4 cell line by blocking the nuclear import of NFAT2. In summary, the platinum treatment plus PARPi or bevacizumab had superiority in extending survival ratios for PSROC patients, but BRCA mutation should be considered. Moreover, the ADP-ribosyl cyclase agonists exhibited improved leukopoiesis, favorable myeloid differentiation, and high immune response, resulting from triggering CD38/ADP-ribosyl cyclase-Ca2+-NFAT signaling axles. This developed CD38 receptor-based drug screening model provided a new approach to high-throughput drug screening for leukopenia.